T3/T7 PRIMER QUESTION!!!

John W. Longshore Gene009 at uabdpo.dpo.uab.edu
Fri Nov 4 09:29:25 EST 1994


In article <stauth-031194111951 at darenmac.usgmrl.ksu.edu>,
stauth at crunch.usgmrl.ksu.edu (Darren Stauth) wrote:

> I'm currently doing universal PCR to amplify and sequence an unknown region
> of DNA.  I've used a couple of different universal primers, both of which
> contain T7 primer sequence but found they caused problems because the TA
> cloning vector also contains a T7 site.  This gives mixed sequence when
> performing sequencing reactions with the T7 primer.  So we changed to a
> uni-primer with a T3 site within it.  By some confusion I used T7 instead
> of T3 in my second round of PCR yet still got amplification.  I assume
> since T7 and T3 are so similar that T7 misannealed to the T3 site.  This
> leads me to my question:  since T7 and T3 are so similar and can misanneal
> to give amplification in PCR, will the same thing happen in sequencing
> reactions?  I've seen vectors containing both T7 and T3 sites.  Has anybody
> had problems sequencing in such a vector?
> 
> -- 
> Darren M. Stauth -- stauth at crunch.usgmrl.ksu.edu

Darren-
   I used T7 and T3 vector primers to cycle sequence clones pulled from a
hippocampal cDNA library with no problems.  I've never had any problems
with misannealing to my knowledge, however, i anneal these primers at 55
to 60 degrees C.

Hope this helps-
                  John

-- 
John W. Longshore
Laboratory of Medical Genetics
University of Alabama at Birmingham
gene009 at uabdpo.dpo.uab.edu



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